A Novel 3D Visualization Method in Mice Identifies the Periportal Lamellar Complex (PLC) as a Key Regulator of Hepatic Ductal and Neuronal Branching Morphogenesis

  1. Tongtong Xu
  2. Fujun Cao
  3. Ruihan Zhou
  4. Qin Chen
  5. Jian Zhong
  6. Yulin Wang
  7. Chaoxin Xiao
  8. Banglei Yin
  9. Chong Chen
  10. Chengjian Zhao

  • Curated by eLife

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    eLife Assessment

    This study identifies the Periportal Lamellar Complex (PLC), an important new structure revealed by a novel 3D imaging method. However, the evidence supporting its distinct cellular identity and functional role is currently incomplete, as it relies on transcriptomic re-analysis and correlation without direct experimental validation. Addressing the key issues of methodological rigor and providing functional evidence is essential to fully substantiate these significant claims.

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Abstract

The liver’s microenvironment consists of interconnected vascular, biliary, and neural networks that regulate homeostasis and disease progression. However, the lack of high-resolution 3D visualization hinders our understanding of their interactions in health and pathology.

In this study, we developed a high-resolution multiplex 3D imaging method integrating multicolor metallic compound nanoparticle (MCNP) perfusion and optimized CUBIC tissue clearing, enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in mouse liver.

Using this approach, we identified a novel perivascular structure, the Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis between its endothelium and the periportal lobule sinusoid. The PLC contains a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, potentially linked to bile acid transport, biliary epithelial cell proliferation, and neural development.

Under normal physiological conditions, the PLC colocalizes with terminal bile duct branches and sympathetic nerve fibers. During liver fibrosis progression, the PLC dynamically extends into liver lobules alongside advancing fibrosis, acting as a scaffold guiding the migration of bile ducts and nerve fibers into the hepatic parenchyma.

Differential gene expression and GO enrichment analyses further revealed neurodevelopmental and axon guidance pathway enrichment in the CD34⁺Sca-1⁺ endothelial population of PLC during fibrosis.

Collectively, these findings establish the PLC as a critical structural and functional hub integrating hepatic vascular, biliary, and neural systems to regulate liver function. This study advances our understanding of liver biology and offers potential therapeutic targets for liver diseases.

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  1. eLife

    eLife Assessment

    This study identifies the Periportal Lamellar Complex (PLC), an important new structure revealed by a novel 3D imaging method. However, the evidence supporting its distinct cellular identity and functional role is currently incomplete, as it relies on transcriptomic re-analysis and correlation without direct experimental validation. Addressing the key issues of methodological rigor and providing functional evidence is essential to fully substantiate these significant claims.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

    Strengths:

    This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

    Weaknesses:

    This manuscript is well-written, organized, and informative. However, there are some points that need to be clarified.

    (1) After MCNP-dye injection, does it remain in the blood vessels, adsorb onto the cell surface, or permeate into the cells? Does the MCNP-dye have cell selectivity?

    (2) All MCNP-dyes were injected after the mice were sacrificed, and the mice's livers were fixed with PFA. After the blood flow had ceased, how did the authors ensure that the MCNP-dyes were fully and uniformly perfused into the microcirculation of the liver?

    (3) It is advisable to present additional 3D perspective views in the article, as the current images exhibit very weak 3D effects. Furthermore, it would be better to supplement with some videos to demonstrate the 3D effects of the stained blood vessels.

    (4) In Figure 1-I, the authors used MCNP-Black to stain the central veins; however, in addition to black, there are also yellow and red stains in the image. The authors need to explain what these stains are in the legend.

    (5) There is a typo in the title of Figure 4F; it should be "stem cell".

    (6) Nuclear staining is necessary in immunofluorescence staining, especially for Figure 5e. This will help readers distinguish whether the green color in the image corresponds to cells or dye deposits.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injecting metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, inferior vena cava, and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers, and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as a scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

    Strengths:

    The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

    Weaknesses:

    This reviewer has several concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results. In this reviewer's opinion, the introduction contains overstatements regarding the potential of the method, there are severe caveats in the method descriptions, and several parts of the Results are not fully supported by the documentation. Thus, the conclusions of the paper may be critically viewed in their present form and may need reconsideration by the authors.

  4. eLife

    Reviewer #3 (Public review):

    Summary:

    In the reviewed manuscript, researchers aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis. The PLC originates from the portal vein and is characterized by a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells. Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting the mRNA presence of genes linked to neurodevelopment, biliary function, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

    The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver at the same time. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists; however, some claims need more thorough assessment by functional experimental approaches to decipher the functional molecules and the sequence of events before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems. Similarly, the level of detail of the methods section does not appear to be sufficient to exactly recapitulate the performed experiments, which is of concern, given that the new technique is a cornerstone of the manuscript.

    Nevertheless, the work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of readers.

    Strengths:

    The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

    This work proposes a new biological framework between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

    Weaknesses:

    Possible overinterpretation of the CD34+Sca1+ findings was built on re-analysis of one scRNAseq dataset.

    Lack of detail in the materials and methods section greatly limits the usefulness of the new technique to other researchers.

  5. Version published to 10.7554/elife.108669.1 on eLife
  6. Version published to 10.7554/elife.108669 on eLife
  7. Version published to 10.1101/2025.08.29.673025 on bioRxiv